- Email: [email protected]
Site specific effects of acute exercise on muscle and adipose tissue metabolism in sedentary female rats
Physiology & Behavior, Vol. 43, pp. 65-71. Copyright©Pergamon Press plc, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
Site Specific Effects of Acute Exercise on Muscle and Adipose Tissue Metabolism in Sedentary Female Rats R. SAVARD, L. J. SMITH, J. E. PALMER AND M. R. C. GREENWOOD 1 V a s s a r College, D e p a r t m e n t o f B i o l o g y , P o u g h k e e p s i e , N Y 12601
Received 24 September 1987 SAVARD, R., L. J. SMITH, J. E. PALMER AND M. R. C. GREENWOOD. Site specific effects of acute exercise on muscle and adipose tissue metabolism in sedentary female rats. PHYSIOL BEHAV 43(1) 65-71, 1988.--The effects of endurance exercise on muscle, and adipose tissue metabolism were investigated. Female lean Zucker rats swam for two hours at high intensity. Three groups were examined: pre-exercise control (C), exercised (E) and 24 hours post-exercise (E-24). Exercise increased fat cell lipolysis in the inguinal depot 09<0.05) while no effect was detected in the parametrial depot. In contrast, parametriai pad lipoprotein lipase (LPL) activity decreased after exercise with 24 hours post-exercise values being reduced below E and C rats (p <0.05). Gastrocnemius LPL activity remained unchanged during exercise while heart LPL increased, E having higher values than C and E-24 (,o<0.05). Gastrocnemius, but not heart, citrate synthase activity increased with exercise, with E-24 values increased compared to E and C (p<0.05). These results demonstrate that adipose tissue's response to exercise is site specific, and suggests a distinct physiological role for different adipose depots. Muscle LPL and citrate synthase activities were modified differently for gastrocnemius and heart, confirming the distinct metabolic response to exercise of these two muscles. Site specificity Fat cell lipolysis
Endurance exercise
Adipose tissue
A M O N G the several behavioral changes known to reduce adiposity, energy expenditure through endurance exercise programs was reported to be successful in man [24,40] and in rats [7] and appeared to induce physiological changes at the level o f muscle and adipose tissue. Overall, muscle glucose and lipid utilization were reported to increase during exercise [46]. Glucose conversion into triglycerides (TG) and lipoprotein lipase (LPL) activity have been shown to decrease in the epididymal fat depot during exercise in trained rats [1,4]. Exercise was reported to increase fat cell lipolysis in intra-abdominal as well as subcutaneous depots [4,33] and this effect may last several hours after one bout of exercise in rats [33]. Because of its physiological interest, the metabolic specificity of adipose tissue from different locations is a phenomenon under increasing investigation [30]. Indeed, site specificity for adipose tissue has been reported for fat cell size and number [5, 29, 41], its lipid composition [32] and also for several metabolic activities [15, 22, 44]. Moreover, site differences in the metabolic response of adipose tissue have also been reported during pregnancy [37,41], menopause [36], and food restriction [3]. Exercise-training is no exception. Although the chronic effects of endurance exercise have been shown to reduce adiposity similarly for different adipose tissue depots in rats [33], site specific responses were reported by others [41]. The relative contribution of
Skeletal muscle
Lipoprotein lipase
different adipose tissue depots in directing substrates to the increased energy requirement of working muscles during exercise is, however, poorly understood. The present study was thus designed to investigate the effects of one bout of high intensity exercise on adipose tissue, and muscle metabolism in female rats; and to elucidate any regional specificity of the responses observed. METHOD Animals All rats used in this study were from the Zucker rat colony established at Vassar College from breeding pairs originally provided by the Harriet G. Bird Memorial Laboratory, Stowe, MA. Virgin female rats (homozygous lean (Fa/Fa) rats) approximately thre e months of age and initially weighing between 175-225 grams were acclimated to the experimental situation before actual testing began. Before and during the acclimation and experimental period, animals were housed in groups of 5 to 7, and maintained on a 12-hour light/12-hour dark cycle (lights on at 06:00). Animals were allowed ad lib access to a standard diet (Agway Lab Chow 3000) and to water throughout the study. Acclimation and Animal Preparation Animals in the present study underwent a period of ac-
1Requests for reprints should be addressed to Dr. M. R. C. Greenwood, Department of Biology, Vassar College, Box 65, Poughkeepsie, NY 12601.
65
66 climation in order to isolate the effects of exercise from other stress associated with swimming in female rats. Thus, all animals used underwent an acclimation period of at least one week of handling and exposure to stimulation of the experimental-day procedure. Between 08:00 and 10:00, animals were removed from the home cage, weighed, and then placed in the swim tank, Idled with two inches of water at 37°C, for approximately one hour. No significant physical activity was caused by this procedure; that is, all animals remained essentially sedentary. After exposure to water for one hour, animals were removed, dried, and carried to the lab. Each rat was then handled and acclimated to the guillotine. This procedure ensured that animals from each group were familiar with most aspects of the protocol except the acute exercise itself. On the day prior to the experimental day, a vaginal smear was taken from each rat and only those scheduled to be in diestrus were selected for use. Thus, all rats ultimately used were in the same phase of their estrus cycle. This procedure controlled for metabolic variations due to differences in circulating reproductive hormone levels. Selected animals were housed individually overnight. Experimental Protocol Our experimental design is consistent with others that have investigated the effects of a single bout of exercise in relation to its energy requirements. Indeed, one bout of high intensity exercise in sedentary rats was reported to modify the lipolytic activity of adipose tissue in a similar manner with swimming [33] or with running on a treadmill [4]. The present experimental design has allowed us to gather information immediately before and after, and 24 hours after one bout of heavy exercise. Rats were divided into three groups: pre-exercise (C), immediately after exercise (E) and 24 hours post-exercise (E-24). C rats (n=8) were placed in two inches of water at 37°C between 07:30 and 08:30. At 08:30, C rats were sacrificed, while the other two groups of rats began the exercise period. Food intake and body weight of all rats were measured throughout study. The exercise program consisted of 2 hours of continuous swimming in 30x 12× 16 inch tanks filled with water at 37°C. No more than four animals swam in the same tank at one time. Two criteria were used to control exercise intensity. First, front and back legs had to work continuously throughout the session. Second, the entire body had to remain under water in such a manner that rats had to work intensively to keep their nose at the surface of the water. After two hours of exercise at this intensity level, animals were near exhaustion, but did not require resuscitation. At 10:30, E rats were removed from the water and sacrificed within five mintutes of the swimming bout. Rats in the post-exercise group (E-24) were removed from the water, dried, and returned to individual cages. Overnight food intake and body weight of E-24 rats were measured and the following day, after exposure to two inches of water at 37°C between 09:00 and 10:30, the animals were killed. All animals were killed by decapitation within five minuted of removal from the home cage or water. Blood collected during decapitation into non-heparinized and heparinized test tubes, centrifuged at 3000×g for fifteen minutes, and stored at -70°C for further glycerol, glucose and corticosterone analyses. Tissue Preparation Since adipose tissue site specific responses have been
SAVARD, SMITH, P A L M E R A N D G R E E N W O O D reported with exercise training [41], two fat pads at different locations were investigated. The inguinal depot was selected at a representative subcutaneous depot while the parametrial fat pad represented deeper fat. Left and right inguinal and parametrial adipose depots were quickly excised and weighed. Tissues for metabolic measures were placed on ice, and a portion was saved for fat cell number and fat cell size determination using the method of Folch et al. [13] and electronic counting of osmium-fixed cells as described by Hirsch and Gallian [19]. Adipose tissue homogenates (1:12) were prepared using ground glass-on-glass homogenizers and 0.25 M sucrose--1 mM EDTA buffer (pH 7.4). Homogenates were centrifuged at 12,000×g for 15 minutes in a Sorvall RC5-B centrifuge refrigerated at 4°C. The postmitochondrial infranatant was aspirated and stored at -70°C for L P L assay. One hundred to 150 mg of parametrial and inguinal adipose tissue was treated with collagenase (Worthington Biochemical Co.) at a low concentration (3.7 mg/ml) in order to minimize any possible damage of the cell membrane as recommended by Gliemann [16]. Tissue was then digested for thirty minutes at 37°C in Krebs-Ringer bicarbonate buffer containing glucose (50 mg/100 ml) and fatty-acid-free bovine serum albumin (4%) (Sigma Chemical Co.) according to the method of Bukowiecki et al. [7]. The isolated fat cells were washed four to five times with fresh buffer and pre-incubated for fifteen minutes at 37°C under an atmosphere of 95% 02/5% COs. An aliquot of final cell suspension was taken for cell viability and cell number determinations, using trypanblue staining, under the light microscope. Isolated, washed fat cells were incubated for thirty minutes at 37°C after oxygenation with 95% 02/5% COs gas, with and without epinephrine for later glycerol determination [7]. Glycerol release of fat cells was performed with epinephrine at 10 ~ M and 10 -:5 M, two levels that represent, in our laboratory, a submaximal and a maximal stimulation of lipolysis. Fat cell lipolysis, determined by this technique, reached a high level of reliability [11] and appeared to follow closely in vivo values of glycerol and unesterified fatty acids ( F F A ) [48]. Indeed, a close parallelism between the effects of epinephrine on glycerol and F F A release from isolated fat cells and incubated tissue and their values in plasma has been reported in lean Zucker rats [48]. Heart and both left and right gastrocnemius muscles were quickly excised and 250-350 mg of tissue were immediately weighed and incubated for heparin release of LPL. Thus, precise horizontal sections of fresh gastrocnemius and heart tissue were prepared in order to measure the overall muscle enzymatic activity. The fresh tissue was cut into 5 to 6 pieces and placed in vials containing 1 ml Krebs-Ringer bicarbonate buffer (pH 7.4) with albumin (4%) and sodium heparin (10 USP units). The tissue was incubated in a shaking water bath for one hour at 37°C. Muscle tissue was removed and the medium was frozen at -70°C for LPL activity determinations. Muscle fresh tissue was rapidly weighed, frozen in liquid nitrogen, and stored at -70°C for later citrate synthase determinations. Assays Lipolysis in isolated fat cells was measured by the amount of glycerol released into the incubation medium [7]. Glycerol was measured fluorometrically by the method of Lowry and Passonneau [28]. Citrate synthase activity, generally recognized as a good indicator of the oxidative capacity of muscles [20], was also measured. Thirty to 40 mg of tissue, obtained
SITE S P E C I F I C E F F E C T S O F A C U T E E X E R C I S E
67
TABLE 1 CHARACTERISTICSOF RATS Mean
Body Weight (g) Food Intake (g) Inguinal Pad Weight (g) Inguinal Cell Size (p.g) Parametrial Pad Weight (g) Parametriai Cell Size 0zg)
C
E
E-24
Differences
214.0 ---20.8 23.3 --- 4.9 0.701_+ 0.118
217.0 _+14.6 21.9 -+ 3.0 0.726_+ 0 . 1 6 8
213.0 ---8.3 21.6 _+2.8 0.637_+0.141
n.s. n.s. n.s.
0.072_+ 0.03
0.068_+ 0.02
0.084_+0.04
n.s.
1.032_+ 0.352
1.030_+ 0 . 2 9 2
1.120_+0.498
n.s.
0.240_+ 0.04
0.213_+ 0.03
0.216_+0.03
n.s.
Values are means_SD, n=8. C: pre-exercise; E: post-exercise; E-24:24 hr post-exercise; n.s.: not significant. A
L P L activity was assayed in the postmitochondrial fraction for adipose tissue and in the heparin released fraction of muscles. The LPL elution was incubated for one hour at 37°C according to a modification [18] of the procedures of Schotz et al. [42]. The latter technique, modified in our laboratories [18], was shown to have a linear reaction during the first 60 min of incubation; the optimal pH being between 7.5 and 8.0. Serum glucose levels were measured on a glucose analyzer (Yellow Springs Instrument Co., Model 27). Corticosterone levels were measured after purification on Celite microcolumns using the chromatographic radioimmunoassay procedures of Wingfield et al. [47]. 14C-corticosterone was purchased from New England Nuclear, Boston, MA. All other enzymes and chemicals used in the assays were purchased from Sigma Chemical Company, St. Louis, MO.
INGUINAL FAT CBLL LIPOLYJlm coo
C r--"'3 •
Statistical Analyses ItlAL
LIPO
The effects of exercise were tested through an analysis of variance and means were compared with a Duncan multiple range test procedure. Differences were considered significant at p<0.05. Correlations were measured with Pearson's correlation coefficient. Because of their physiological interest, correlations between variables that were significantly modified by exercise were calculated for the pre- and postexercise values and were considered significant at p<0.05. Results
BIgAL
I~°M
10~U
FIG. 1. Inguinal (A) and parametrial (B) fat cell glycerol release for its basal, submaximal (10-5 M) and maximal (10-3 M) epinephrine stimulated values (mean- S.E.). C= non-exercised rats; E = data collected immediately after exercise; E-24=data collected 24 hours after exercise. *p<0.05, n=8. from a precise horizontal section of muscle, were thawed, minced and homogenized in ice-cold solution of 175 mM KCI with 10 mM glutathione and 2 mM EDTA at pH 7.4 using glass-on-glass homogenizers. Homogenates were frozen and thawed three times to disrupt the mitchondria prior to the enzyme assay. Citrate synthase was then assayed at 30°C by the method of Srere [43] using 5,5-dithiobis-(2-nitrobenzate).
Body weight, food intake, adipose tissue weights and fat cell size in the inguinal and parametrial depots were similar for the three groups (Table 1). Acute exercise increased fat cell lipolysis for the inguinal depot but not the parametrial depot (Fig. 1). Both tissues responded significantly to the stimulation with epinephrine (o<0.05). Basal, submaximal and maximal epinephrine stimulated values were higher in the exercise group (E) than controis for the inguinal adipose tissue (p <0.05) while no significant changes were observed in the parametrial depot. Adipose tissue L P L activity decreased following exercise in the parametrial depot with E-24 values significantly smaller than C and E values (0<0.05) (Fig. 2). Adipose tissue L P L in the inguinal depot did not change with exercise (Fig. 2). On the other hand, exercise increased heart L P L activity in the E group (0<0.05) but did not affect L P L activity in the gastrocnemius (Fig. 3).
68
SAVARD, SMITH, PALMER AND GREENWOOD
Lipoprotein Lipase
Lipoprotein Lipase I
O
gl °~ 4~
O
O
C
1~
g-24
INGUINAL
C
g
g-24
PARAMETRIAL
C
g
g-24
GABTROCNRMIU8
1
g
g-'m4
HEART
FIG. 2. Inguinal and parametrial post-mitochondrial adipose tissue lipoprotein lipase activity (mean+-S.E.). C=non-exercised rats; E=data collected immediately after exercise; E-24=data collected 24 hours after exercise. *p<0.05, n=8.
FIG. 3. Gastrocnemius and heart muscle heparin released lipoprotein lipase activity (mean_+S.E.). C=non-exercised rats; E=data collected immediately after exercise; E-24=data collected 24 hours after exercise. *p<0.05, n=8.
Gastrocnemius citrate synthase remained unchanged immediately after exercise but reached values significantly higher 24 hours later (p<0.05) (Fig 4). Heart citrate synthase values are not significantly changed with exercise (Fig. 4). Blood glycerol and blood glucose increased after exercise, with E greater than C and E-24 post-exercise (p<0.05). No change was observed for the plasma glucocorticoid levels (Table 2).
while no effects of exercise were apparent for parametrial fat cell lipolysis, its LPL activity was gradually suppressed in a manner similar to that reported by Barakat et al. [4]; the lowest values being observed 24 hours after the bout of exercise. The present results are thus consistent with the interpretation that one bout of high intensity exercise has site specific effects on adipose tissue metabolism. Moreover, parametrial adipose tissue LPL activity reached values about 4 fold higher than in the inguinal depot. This finding has also been reported by others [17] and stresses the possibility that each tissue has a specific physiological role. The latter specificity has also been observed during chronic exercisetraining. Indeed, recent results from our laboratory have confirmed that parametrial and retroperitoneal adipose tissues exhibit a greater metabolic response to exercise training during pregnancy than does the inguinal depot [41]. The present results appeared to be independent of food intake. For example, while adipose tissue LPL activity was decreased 24 hr after exercise, exercised and non-exercised animals had similar food intake. Similar results were also reported for the epididymal fat depot of Holtzman [4] and Wistar rats [38]. The mechanism responsible for the regional specificity observed in the response to one bout of exercise in adipose tissue lipolytic and LPL activities could occur with the action of the sympathetic innervation and blood flow. At the level of adipose tissue, a sympathetic innervation is first known to induce a local vasoconstriction along with a decreased metabolic activity; second, an accumulation of adenosine in adipose tissue; and third, a local vasodilatation particularly strong when the stimuli is intense [14,31]. Pefonnet et al. [34,35] reported that the local sympathetic stimulation is mainly responsible for the metabolic changes of adipose tis-
Metabolic Relationships Within C and E Groups The metabolic relationships between pre- and postexercise values for gastrocnemius citrate synthase activity and heart and parametrial L P L are markedly modified by acute exercise (Table 3). Gastrocnemius citrate synthase activity is negatively correlated with heart L P L before exercise and positively afterward (,o<0.05). On the other hand, it is positively correlated with parametrial L P L activity before and negatively after the exercise bout (p<0.05). DISCUSSION
Overall Effects of Exercise Results of the present study showed clearcut differences in the metabolic responses to one bout of high intensity exercise for adipose tissues of deep and subcutaneous anatomical regions. In agreement with the results reported by Oscai [33], inguinal fat cell lipolysis was higher at the end of one bout of swimming than at the beginning, values tending to come back to control 24 hours later. Plasma glycerol levels were concordant with the latter finding, i.e., values were increased after exercise and were back to pre-exercise levels 24 hours later. Inguinal adipose tissue LPL activity was however unchanged by exercise. On the other hand,
SITE S P E C I F I C E F F E C T S O F A C U T E E X E R C I S E
69 TABLE 2
EFFECTS OF EXERCISE ON BLOOD GLYCEROL, GLUCOSE AND CORTICOSTERONE Mean
C Plasma Glycerol (ttmol/dl) Serum Glucose (mg/dl) Plasma Corticosterone (ng/ml)
E
13.48-+ 2.77
19.07-+1 . 8 0
E-24
Differences
15.32-+2.88
*
131.8 -+13.2
212.7 -+76.3
133.4 -+10.8
*
170.8 -+33.3
172.9 -+50.9
194.3 -+55.4
n.s.
Values are means-+SD, n=8. C: pre-exercise; E: post-exercise; E-24:24 hr post-exercise. *E Significantly different than C and E-24 (p<0.05). n.s.: not significant. 90"
0
60 \\\\ \\\N \\\\
*d
.
30
!
! !
\\ \\ \\ N \
r..)
C
c
E
E-24
GASTROCNEMIUS
C
E
E-24
HEART
FIG. 4. Gastrocnemius and heart muscle citrate synthase activity (mean- S.E.). C = non-exercised rats; E = data coUected immediately after exercise; E-24=data collected 24 hours after exercise. *p <0.05, n=8. sue during exercise. Moreover, changes in adipose tissue blood flow during exercise are generally reported to parallel changes of its metabolism [8,9]. The latter steps of action of the sympathetic innervation could however vary with the site studied [2]. A stimuli such as exercise was reported to reduce splanchnic blood flow [39] while it increases blood flow o f subcutaneous adipose tissue as well as its lipolytic activity [10]. This observation along with the previous results indi-
cate that the sympathetic stimulation and its consequences on blood flow could not necessarily be homogeneous. Since adipose tissue is largely infiltrated by sympathetic nerve endings, differences in the metabolic responses observed in the present study could, at least partly, happen through the heterogeneity of the sympathetic nerve stimulation. The latter stimulation could induce changes in blood flow as well as local concentrations of several metabolites such as adenosine and fatty acids known to modify metabolic activities of adipose tissue. Fat losses in exercise-trained rats were reported to be similar in both the subcutaneous and the intra-abdominal adipose depot [33]. The present results, i.e., the increased lipolytic activity in the subcutaneous depot and the decreased L P L activity in the intra-abdominal depot after exercise suggest that reduction of adiposity with exercisetraining could happen through different metabolic pathways. Moreover, the lack of a relationship between individual adipose tissue L P L and lipolytic activities before and after exercise suggests that these regulatory phenomena may be independently controlled and not reciprocally related in any stoichiometric way. The muscle metabolic responses to exercise were also different for the two tissues studied. L P L activity increased only for the heart muscle, a result similar to the results reported by Oscai [33]. This finding supports the concept that heart is an important site of intra-cellular oxidation of fatty acids derived from increased lipolysis and heart LPL. The latter oxidative process is greatly involved in the energy needs of heart muscle of rodents since their cardiac myosin ATPase was reported to be much higher than other species [27]. Citrate synthase activity, on the other hand, was responsive only in the gastrocnemius muscle, its activity having a 30% increase 24 hours after the bout of exercise. Similarly, Dohm et al. [12] reported no change of skeletal muscle citrate synthase activity in rats immediately after running until exhaustion. No values were reported within the several hours following the test [12]. It is known, however, that the mitochondrial content of skeletal muscles increases rapidly in response to exercise [6]. Indeed, Booth and Holloszy [6] reported a detectable increase of plantaris citrate synthase activity as soon as 2 days after the beginning of an exercise program on treadmill in rats. These results and ours suggest that the oxidative capacity of skeletal muscle can rapidly respond to exercise when the latter is heavy work load that
70
SAVARD, SMITH, PALMER AND GREENWOOD
TABLE 3 INTERCLASSCORRELATIONSBETWEENGASTROCNEMIUS CITRATESYNTHASEACTIVITYAND HEART AND PARAMETRIAL LPL ACTIVITIES Gast. Citrate Synthase
Heart LPL Parametrial LPL
Pre-Exercise
Post-Exercise
-0.77* 0.70*
0.74* -0.72*
*p~<0.05. stresses the synthesis of the enzyme by muscle cells [6]. While the absolute amount of work performed by each rat could not be directly measured in the present study, the level of intensity of exercise was substantial. Indeed, swimming at a non-exhaustive level has been reported to at least double the oxygen consumption in rats [23]. The present swimming protocol was designed to exercise animals continuously at high intensity for two hours. Increased blood glucose concentrations such as reported in this study are usually related to heavy exercise and are thought to originate mainly from an increased glucose output from the liver [45]. In a similar fashion, Jones et al. [21] reported increased plasma glucose during heavy exercise in humans while no such observation was made with light exercise. One ought however to take into account the delay between the end of exercise and the time of sacrifice (less than 5 min); time during which liver gluconeogenesis is elevated in resting animals and contributes to increase furthermore glucose levels into the blood. Pre- and Post-Exercise Correlations
Correlations between variables that were modified by the bout of high intensity exercise were compared before and after swimming. This procedure allows us to verify qualitatively and quantitatively if the changes induced by exercise, modified the relationships between the variables. This approach, although not necessarily indicating causality, helps to clarify the manner by which each matabolic pathway is modified. Animals in a rested fed state are primarily dependent upon lipoproteins and glucose as substrates for the basal metabolic activity of tissues. The positive pre-exercise corre-
lation between gastrocnemius citrate synthase activity and parametrial LPL activity suggests that the skeletal muscle glycolytic fibers are using mainly glucose for their energetic needs while, through adipose tissue LPL, lipids are stored in the adipose tissue. Consistent with high availability of circulating substrates during rest, heart LPL is low. This low activity may be mediated by the high insulin: glucagon ratio which is characteristic of the fed state, and is thought to inhibit muscle LPL acitivity [25,26] while stimulating adipose tissue LPL. These relationships were rapidly modified after exercise. The negative correlation between post-exercise gastrocnemius citrate synthase and parametrial LPL is consistent with the interpretation that low values of adipose tissue LPL activity during exercise are related to a high oxidative metabolism of skeletal muscles, suggesting high efficiency in directing energy fuels to the working muscles. In addition, the positive correlation of gastrocnemius citrate synthase with heart LPL after exercise strengthen the interpretation that both metabolic pathways are involved in the specific supply of energy of these two muscles during exercise. One should thus expect that the latter high efficiency reached by some rats of the present study should, for others, be part of their chronic adaptation to endurance exercise. In conclusion, one bout of intense exercise appears to modify the metabolism of energy substrates in a regionally specific manner. Indeed, intra-abdominal adipose tissue metabolism responded differently to exercise than subcutaneous adipose tissue suggestive of a distinct physiological role for each adipose depot. Moreover, skeletal and heart muscle were also specifically responsive to endurance exercise. LPL activity was mainly affected in the heart while the citrate synthase responded more in the gastrocnemius muscle. ACKNOWLEDGEMENTS The authors gratefully acknowledge the expert technical assistance of Jerry Calvin and Jim Brown. We thank Dr. Marilyn Ramenofsky for determining blood corticosterone levels. We also thank Ms. Bonnie Milne from Vassar College and secretary's staff of the Department of Physical Education, University of Montreal for their typing assistance. Comments on the manuscript from Dr. Ruth Kava and Dr. David West were also very helpful. This study was supported by National Institute of Child Health and Human Development Grant HD-12637, and by the Fonds de la Recherche en Sant6 du Qurbec 830540, 840043, 850471.
REFERENCES
1. Askew, E. W., H. Barakat, C. L. Kuhl and G. L. Dohm. Response of lipogenesis and fatty acid synthetase to physical training and exhaustive exercise in rats. Lipids 10: 491-496, 1975. 2. Ballard, K. and S. Rosells. Adrenergic neurohumoral influences on circulation and lipolysis in canine omental adipose tissue. Circ Res 28: 389-395, 1971. 3. Baker, N., D. B. Learn, R. Kannan and K. R. Bruckdorfer. Comparison of lipogenic responses to dietary glucose in selected mouse adipose tissues. Ann Nutr Metal 25: 245-254, 1981. 4. Barakat, H. A., D. S. Kerr, E. B. Tapscott and G. L. Dohm. Changes in plasma lipids and lipolytic activity during recovery from exercise of untrained rats. Proc Soc Exp Biol Med 166: 162-166, 1981. 5. Bonnet, F. P., D. Rocour-Brumioul and A. Heuskin. Regional variations of adipose cell size and local cellularity in human subcutaneous fat during normal cell growth. Acta Paediatr Belg 32: 17-27, 1979.
6. Booth, F. W. and J. O. Holloszy. Cytochrome c turnover in rat skeletal muscle. J Biol Chem 252: 416-419, 1977. 7. Bukowiecki, L., J. Lupien, N. Follea, A. Paradis, D. Richard and J. Leblanc. Mechanism of enhanced lipolysis in adipose tissue of exercise-trained rats. Am J Physiol 239: E422-E429, 1980. 8. Bulow, J. Subcutaneous adipose tissue blood flow and triacyiglycerol mobilization during prolonged exercise in dogs. Pflugers Arch 392: 230-234, 1982. 9. Bulow, J. and J. Madsen. Influence of blood flow on fatty acid mobilization from lipolytically active adipose tissue. Pflugers Arch 390: 169-174, 1981. 10. Bulow, J. and E. Tondevold. Blood flow in different adipose tissue depots during prolonged exercise in dogs. Pflugers Arch 392: 235-238, 1982. 1I. Despres, J. P., C. Bouchard, L. Bukowiecki, R. Savard and J. Lupien. Morphology and metabolism of human fat cells: a reliability study, lnt J Obes 7: 231-240, 1983.
SITE SPECIFIC EFFECTS OF ACUTE EXERCISE
12. Dohm, G. L., R. L. Huston, E. W. Askew and H. L. Fleshood. Effects of exercise training, and diet on muscle citric acid cycle enzyme activity. Can J Biochem 51: 849--854, 1973. 13. Folch, J., M. Lees and G. H. Sloane Stanley. A simple method for the isolation and purification of total lipid from animal tissues. J Biol Chem 226: 497-509, 1957. 14. Fredholm, B. B.Studies on the sympathetic regulation of circulation and metabolism in isolated subcutaneous adipose tissue. Acta Physiol Scan Suppl 354: 1-47, 1970. 15. Fried, S., M. Lavau and F. X. Pi-Sunyer. Variations in glucose metabolism by fat cells from three adipose depots of the rat. Metabolism 31: 876--883, 1982. 16. Gliemann, J. Assay of insulin-like activity by the isolated fat cell method. Factors influencing the response to crystalline insulin. Diabetologia 3: 382-388, 1967. 17. Gruen, R. K. amd M. R. C. Greenwood. Adipose tissue iipoprotein lipase and glycerol release in fasted Zucker (fa/fa) rats. Am J Physiol 4: E76--E83, 1981. 18. Hietanen, E. and M. R.C. Greenwood. A comparison of lipoprotein lipase acitvity and adipocyte differenciation in growing male rats. J Lipid Res 18: 480-490, 1977. 19. Hirsch, J. and E. Gallian. Methods for the determination of adipose cell size in man and animals. J Lipid Res 9:110-119, 1968. 20. Holloszy, J. O. and F. W. Booth. Biochemical adaptations to endurance exercise in muscle. Annu Rev Physiol 38: 273-291, 1976. 21. Jones, N. J., G. J. F. Heigenhauser, A. Kuksis, C. G. Matsos, J. R. Sutton and C. J. Toews. Fat metabolism in heavy exercise. Clin Sci 59: 469-478, 1980. 22. Kannan, R. and N. Baker. Lipogenic responses to dietary glucose in selected rat adipose tissues. Indian J Biochem Biophys 18: 47-50, 1981. 23. Leblanc, J., A. Labrie, D. Lupien and D. Richard. Catecholamines and triiodo-thyronine variations and the calorigenic responses to norepinephrine in cold-adapted and exercise-trained rats. Can J Physiol Pharmacol 60: 783-787, 1982. 24. Leon, A. S., J, Conrad, D. B. Hunninghake and R. Serfass. Effects of a vigorous walking program on body composition, and carbohydrate and lipid metabolism of obese young men. Am J Clin Nutr 33: 1776-1787, 1979. 25. Lithell, H., J. Boberg, K. Hellsing, G. Lundqvist and B. Vessby. Lipoprotein-lipase activity in human skeletal muscle and adipose tissue in the fasting and fed states. Atherosclerosis 30: 89-94, 1978. 26. Lithell, H., J. Boberg, K. Hellsing, G. Lundqvist and B. Vessby. Increase of the lipoprotein-lipase activity in human skeletal muscle during clofibrate administration. Eur J Clin Invest 8: 6774, 1978. 27. Lompre, A. M., J. J. Mercadier, C. Wisnewsky, P. Bouveret, C. Pantanoli, A. D'Albis and K. Schwartz. Species- and agedependent changes in the relative amounts of cardiac myosin isoenzymes in mammals. Dev Biol 84: 286--290, 1981. 28. Lowry, O. and J. A. Passonneau. Flexible System o f Enzymatic Analysis. New York: Academic, 1972. 29. Meade, C. J. and M. Ashwell. Site differences in fat cells of New Zealand obese mice--a transplantation study. Metabolism 29: 854-858, 1980. 30. Mrosovsky, N. Body fat: What is regulated? Physiol Behav 38: 407-414, 1986.
71
31. Ngai, S. H., S. Rosell and L. Wallenberg. Nervous regulation of blood flow in the subcutaneous adipose tissue in dogs. Acta Physiol Scand 68: 397-403, 1966. 32. Pittet, P. G., D. Halliday and P. E. Bateman. Site differences in the fatty acid composition of subcutaneous adipose tissue of obese women. Br J Nutr 42: 57-60, 1979. 33. Oscai, L. B. Effect of acute exercise on tissue free fatty acids in untrained rats. Can J Physiol Pharmacol 57: 485-489, 1979. 34. Prronnet, F. and A. Imbach. Endurance and metabolic adjustments to exercise in sympathectomized (6-OHDA) rats. In: Biochemistry o f Exercise, vol 13, edited by H. G. Knuttgen, J. A. Vogel and J. Poortmans. Champaign: Human Kinetics Publishers, 1983, pp. 762-765. 35. Prronnet, F., R. Nadeau, J. de Champlain and A. Imbach. Plasma catecholamines and reponse to exercise in 6hydroxydopamine-treated dogs. Can J Physiol Pharmacol 60: 1219-1224, 1982. 36. Rebuffr-Scrive, M., J. Eldh, L. O. Hafstrom and P. Bjorntorp. Metabolism of mammary, abdominal, and femoral adipocytes in women before and after menopause. Metabolism 35: 792-797, 1986. 37. Rebuffr-Scrive, M., L. Enk, N. Crona, P. Lonnroth, L. Abrahamsson, U. Smith and P. Bjorntorp. Fat cell metabolism in different regions in women. Effect on menstrual cycle, pregnancy and lactation. J Clin Invest 75: 1-4, 1985. 38. Ranramaa, R. Acute effect of physical exercise on glycogen content and lipoprotein lipase activity in untrained diabetic rats. Med Biol 60: 139-143, 1982. 39. Rowell, L. B. Human cardiovascular adjustments to exercise and thermal stress. Physiol Rev 54: 75-159, 1974. 40. Savard, R., J. P. Desprrs, M. Marcotte and C. Bouchard. Endurance training and glucose conversion into triglycerides in human fat cells. J Appl Physiol 58: 230-235, 1985. 41. Savard, R., J. E. Palmer and M. R. C. Greenwood. The effects of exercise training on regional adipose tissue metabolism in pregnant rats. Am J Physiol 250: R837-R844, 1986. 42. Schotz, M., A. Garfinkel, J. Huebotter and J. E. Stewart. A rapid assay for lipoprotein lipase activity. J Lipid Res 28:25-31, 1969. 43. Stere, P. A. Methods ofEnzymology, vol 13, edited by J. M. Lowenstein. New York: Academic Press, 1969, p. 34. 44. Tshuchimoto, M., Y. Tokumitsu and M. Ui. Comparison of basal metabolic activities and hormonal sensitivities between mesenteric and epididymal adipocytes. Comp Biochem Physiol 82A: 377-383, 1985. 45. Wahren, J., P. Felig, G. Ahlborg and L. Jorfeldt. Glucose metabolism during leg exercise in man. J Clin Invest 50: 2715-2725, 1971. 46. Wahren, J., P. Felig, L. Hagenfeldt, R. Hendler and G. Ahlborg. Splanchnic and leg metabolism of glucose, free fatty acids and amino acids during prolonged exercise in man: Metabolic adaptation to prolonged exercise. Second International Symposium on Biochemistry of Exercise, edited by H. Howald and J. R. Poortmans. Basel: Magglingen, Birkhauser Verlag, 1973, pp. 144-153. 47. Wingfield, J. C. and D. S. Farner. The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding. Steroids 26:311-327, 1975. 48. Zucker, L. M. Fat mobilization in vitro and in vivo in the genetically obese Zucker rat "fatty." J Lipid Res 13: 234-243, 1972.
0031-9384/88 $3.00 + .00
Site Specific Effects of Acute Exercise on Muscle and Adipose Tissue Metabolism in Sedentary Female Rats R. SAVARD, L. J. SMITH, J. E. PALMER AND M. R. C. GREENWOOD 1 V a s s a r College, D e p a r t m e n t o f B i o l o g y , P o u g h k e e p s i e , N Y 12601
Received 24 September 1987 SAVARD, R., L. J. SMITH, J. E. PALMER AND M. R. C. GREENWOOD. Site specific effects of acute exercise on muscle and adipose tissue metabolism in sedentary female rats. PHYSIOL BEHAV 43(1) 65-71, 1988.--The effects of endurance exercise on muscle, and adipose tissue metabolism were investigated. Female lean Zucker rats swam for two hours at high intensity. Three groups were examined: pre-exercise control (C), exercised (E) and 24 hours post-exercise (E-24). Exercise increased fat cell lipolysis in the inguinal depot 09<0.05) while no effect was detected in the parametrial depot. In contrast, parametriai pad lipoprotein lipase (LPL) activity decreased after exercise with 24 hours post-exercise values being reduced below E and C rats (p <0.05). Gastrocnemius LPL activity remained unchanged during exercise while heart LPL increased, E having higher values than C and E-24 (,o<0.05). Gastrocnemius, but not heart, citrate synthase activity increased with exercise, with E-24 values increased compared to E and C (p<0.05). These results demonstrate that adipose tissue's response to exercise is site specific, and suggests a distinct physiological role for different adipose depots. Muscle LPL and citrate synthase activities were modified differently for gastrocnemius and heart, confirming the distinct metabolic response to exercise of these two muscles. Site specificity Fat cell lipolysis
Endurance exercise
Adipose tissue
A M O N G the several behavioral changes known to reduce adiposity, energy expenditure through endurance exercise programs was reported to be successful in man [24,40] and in rats [7] and appeared to induce physiological changes at the level o f muscle and adipose tissue. Overall, muscle glucose and lipid utilization were reported to increase during exercise [46]. Glucose conversion into triglycerides (TG) and lipoprotein lipase (LPL) activity have been shown to decrease in the epididymal fat depot during exercise in trained rats [1,4]. Exercise was reported to increase fat cell lipolysis in intra-abdominal as well as subcutaneous depots [4,33] and this effect may last several hours after one bout of exercise in rats [33]. Because of its physiological interest, the metabolic specificity of adipose tissue from different locations is a phenomenon under increasing investigation [30]. Indeed, site specificity for adipose tissue has been reported for fat cell size and number [5, 29, 41], its lipid composition [32] and also for several metabolic activities [15, 22, 44]. Moreover, site differences in the metabolic response of adipose tissue have also been reported during pregnancy [37,41], menopause [36], and food restriction [3]. Exercise-training is no exception. Although the chronic effects of endurance exercise have been shown to reduce adiposity similarly for different adipose tissue depots in rats [33], site specific responses were reported by others [41]. The relative contribution of
Skeletal muscle
Lipoprotein lipase
different adipose tissue depots in directing substrates to the increased energy requirement of working muscles during exercise is, however, poorly understood. The present study was thus designed to investigate the effects of one bout of high intensity exercise on adipose tissue, and muscle metabolism in female rats; and to elucidate any regional specificity of the responses observed. METHOD Animals All rats used in this study were from the Zucker rat colony established at Vassar College from breeding pairs originally provided by the Harriet G. Bird Memorial Laboratory, Stowe, MA. Virgin female rats (homozygous lean (Fa/Fa) rats) approximately thre e months of age and initially weighing between 175-225 grams were acclimated to the experimental situation before actual testing began. Before and during the acclimation and experimental period, animals were housed in groups of 5 to 7, and maintained on a 12-hour light/12-hour dark cycle (lights on at 06:00). Animals were allowed ad lib access to a standard diet (Agway Lab Chow 3000) and to water throughout the study. Acclimation and Animal Preparation Animals in the present study underwent a period of ac-
1Requests for reprints should be addressed to Dr. M. R. C. Greenwood, Department of Biology, Vassar College, Box 65, Poughkeepsie, NY 12601.
65
66 climation in order to isolate the effects of exercise from other stress associated with swimming in female rats. Thus, all animals used underwent an acclimation period of at least one week of handling and exposure to stimulation of the experimental-day procedure. Between 08:00 and 10:00, animals were removed from the home cage, weighed, and then placed in the swim tank, Idled with two inches of water at 37°C, for approximately one hour. No significant physical activity was caused by this procedure; that is, all animals remained essentially sedentary. After exposure to water for one hour, animals were removed, dried, and carried to the lab. Each rat was then handled and acclimated to the guillotine. This procedure ensured that animals from each group were familiar with most aspects of the protocol except the acute exercise itself. On the day prior to the experimental day, a vaginal smear was taken from each rat and only those scheduled to be in diestrus were selected for use. Thus, all rats ultimately used were in the same phase of their estrus cycle. This procedure controlled for metabolic variations due to differences in circulating reproductive hormone levels. Selected animals were housed individually overnight. Experimental Protocol Our experimental design is consistent with others that have investigated the effects of a single bout of exercise in relation to its energy requirements. Indeed, one bout of high intensity exercise in sedentary rats was reported to modify the lipolytic activity of adipose tissue in a similar manner with swimming [33] or with running on a treadmill [4]. The present experimental design has allowed us to gather information immediately before and after, and 24 hours after one bout of heavy exercise. Rats were divided into three groups: pre-exercise (C), immediately after exercise (E) and 24 hours post-exercise (E-24). C rats (n=8) were placed in two inches of water at 37°C between 07:30 and 08:30. At 08:30, C rats were sacrificed, while the other two groups of rats began the exercise period. Food intake and body weight of all rats were measured throughout study. The exercise program consisted of 2 hours of continuous swimming in 30x 12× 16 inch tanks filled with water at 37°C. No more than four animals swam in the same tank at one time. Two criteria were used to control exercise intensity. First, front and back legs had to work continuously throughout the session. Second, the entire body had to remain under water in such a manner that rats had to work intensively to keep their nose at the surface of the water. After two hours of exercise at this intensity level, animals were near exhaustion, but did not require resuscitation. At 10:30, E rats were removed from the water and sacrificed within five mintutes of the swimming bout. Rats in the post-exercise group (E-24) were removed from the water, dried, and returned to individual cages. Overnight food intake and body weight of E-24 rats were measured and the following day, after exposure to two inches of water at 37°C between 09:00 and 10:30, the animals were killed. All animals were killed by decapitation within five minuted of removal from the home cage or water. Blood collected during decapitation into non-heparinized and heparinized test tubes, centrifuged at 3000×g for fifteen minutes, and stored at -70°C for further glycerol, glucose and corticosterone analyses. Tissue Preparation Since adipose tissue site specific responses have been
SAVARD, SMITH, P A L M E R A N D G R E E N W O O D reported with exercise training [41], two fat pads at different locations were investigated. The inguinal depot was selected at a representative subcutaneous depot while the parametrial fat pad represented deeper fat. Left and right inguinal and parametrial adipose depots were quickly excised and weighed. Tissues for metabolic measures were placed on ice, and a portion was saved for fat cell number and fat cell size determination using the method of Folch et al. [13] and electronic counting of osmium-fixed cells as described by Hirsch and Gallian [19]. Adipose tissue homogenates (1:12) were prepared using ground glass-on-glass homogenizers and 0.25 M sucrose--1 mM EDTA buffer (pH 7.4). Homogenates were centrifuged at 12,000×g for 15 minutes in a Sorvall RC5-B centrifuge refrigerated at 4°C. The postmitochondrial infranatant was aspirated and stored at -70°C for L P L assay. One hundred to 150 mg of parametrial and inguinal adipose tissue was treated with collagenase (Worthington Biochemical Co.) at a low concentration (3.7 mg/ml) in order to minimize any possible damage of the cell membrane as recommended by Gliemann [16]. Tissue was then digested for thirty minutes at 37°C in Krebs-Ringer bicarbonate buffer containing glucose (50 mg/100 ml) and fatty-acid-free bovine serum albumin (4%) (Sigma Chemical Co.) according to the method of Bukowiecki et al. [7]. The isolated fat cells were washed four to five times with fresh buffer and pre-incubated for fifteen minutes at 37°C under an atmosphere of 95% 02/5% COs. An aliquot of final cell suspension was taken for cell viability and cell number determinations, using trypanblue staining, under the light microscope. Isolated, washed fat cells were incubated for thirty minutes at 37°C after oxygenation with 95% 02/5% COs gas, with and without epinephrine for later glycerol determination [7]. Glycerol release of fat cells was performed with epinephrine at 10 ~ M and 10 -:5 M, two levels that represent, in our laboratory, a submaximal and a maximal stimulation of lipolysis. Fat cell lipolysis, determined by this technique, reached a high level of reliability [11] and appeared to follow closely in vivo values of glycerol and unesterified fatty acids ( F F A ) [48]. Indeed, a close parallelism between the effects of epinephrine on glycerol and F F A release from isolated fat cells and incubated tissue and their values in plasma has been reported in lean Zucker rats [48]. Heart and both left and right gastrocnemius muscles were quickly excised and 250-350 mg of tissue were immediately weighed and incubated for heparin release of LPL. Thus, precise horizontal sections of fresh gastrocnemius and heart tissue were prepared in order to measure the overall muscle enzymatic activity. The fresh tissue was cut into 5 to 6 pieces and placed in vials containing 1 ml Krebs-Ringer bicarbonate buffer (pH 7.4) with albumin (4%) and sodium heparin (10 USP units). The tissue was incubated in a shaking water bath for one hour at 37°C. Muscle tissue was removed and the medium was frozen at -70°C for LPL activity determinations. Muscle fresh tissue was rapidly weighed, frozen in liquid nitrogen, and stored at -70°C for later citrate synthase determinations. Assays Lipolysis in isolated fat cells was measured by the amount of glycerol released into the incubation medium [7]. Glycerol was measured fluorometrically by the method of Lowry and Passonneau [28]. Citrate synthase activity, generally recognized as a good indicator of the oxidative capacity of muscles [20], was also measured. Thirty to 40 mg of tissue, obtained
SITE S P E C I F I C E F F E C T S O F A C U T E E X E R C I S E
67
TABLE 1 CHARACTERISTICSOF RATS Mean
Body Weight (g) Food Intake (g) Inguinal Pad Weight (g) Inguinal Cell Size (p.g) Parametrial Pad Weight (g) Parametriai Cell Size 0zg)
C
E
E-24
Differences
214.0 ---20.8 23.3 --- 4.9 0.701_+ 0.118
217.0 _+14.6 21.9 -+ 3.0 0.726_+ 0 . 1 6 8
213.0 ---8.3 21.6 _+2.8 0.637_+0.141
n.s. n.s. n.s.
0.072_+ 0.03
0.068_+ 0.02
0.084_+0.04
n.s.
1.032_+ 0.352
1.030_+ 0 . 2 9 2
1.120_+0.498
n.s.
0.240_+ 0.04
0.213_+ 0.03
0.216_+0.03
n.s.
Values are means_SD, n=8. C: pre-exercise; E: post-exercise; E-24:24 hr post-exercise; n.s.: not significant. A
L P L activity was assayed in the postmitochondrial fraction for adipose tissue and in the heparin released fraction of muscles. The LPL elution was incubated for one hour at 37°C according to a modification [18] of the procedures of Schotz et al. [42]. The latter technique, modified in our laboratories [18], was shown to have a linear reaction during the first 60 min of incubation; the optimal pH being between 7.5 and 8.0. Serum glucose levels were measured on a glucose analyzer (Yellow Springs Instrument Co., Model 27). Corticosterone levels were measured after purification on Celite microcolumns using the chromatographic radioimmunoassay procedures of Wingfield et al. [47]. 14C-corticosterone was purchased from New England Nuclear, Boston, MA. All other enzymes and chemicals used in the assays were purchased from Sigma Chemical Company, St. Louis, MO.
INGUINAL FAT CBLL LIPOLYJlm coo
C r--"'3 •
Statistical Analyses ItlAL
LIPO
The effects of exercise were tested through an analysis of variance and means were compared with a Duncan multiple range test procedure. Differences were considered significant at p<0.05. Correlations were measured with Pearson's correlation coefficient. Because of their physiological interest, correlations between variables that were significantly modified by exercise were calculated for the pre- and postexercise values and were considered significant at p<0.05. Results
BIgAL
I~°M
10~U
FIG. 1. Inguinal (A) and parametrial (B) fat cell glycerol release for its basal, submaximal (10-5 M) and maximal (10-3 M) epinephrine stimulated values (mean- S.E.). C= non-exercised rats; E = data collected immediately after exercise; E-24=data collected 24 hours after exercise. *p<0.05, n=8. from a precise horizontal section of muscle, were thawed, minced and homogenized in ice-cold solution of 175 mM KCI with 10 mM glutathione and 2 mM EDTA at pH 7.4 using glass-on-glass homogenizers. Homogenates were frozen and thawed three times to disrupt the mitchondria prior to the enzyme assay. Citrate synthase was then assayed at 30°C by the method of Srere [43] using 5,5-dithiobis-(2-nitrobenzate).
Body weight, food intake, adipose tissue weights and fat cell size in the inguinal and parametrial depots were similar for the three groups (Table 1). Acute exercise increased fat cell lipolysis for the inguinal depot but not the parametrial depot (Fig. 1). Both tissues responded significantly to the stimulation with epinephrine (o<0.05). Basal, submaximal and maximal epinephrine stimulated values were higher in the exercise group (E) than controis for the inguinal adipose tissue (p <0.05) while no significant changes were observed in the parametrial depot. Adipose tissue L P L activity decreased following exercise in the parametrial depot with E-24 values significantly smaller than C and E values (0<0.05) (Fig. 2). Adipose tissue L P L in the inguinal depot did not change with exercise (Fig. 2). On the other hand, exercise increased heart L P L activity in the E group (0<0.05) but did not affect L P L activity in the gastrocnemius (Fig. 3).
68
SAVARD, SMITH, PALMER AND GREENWOOD
Lipoprotein Lipase
Lipoprotein Lipase I
O
gl °~ 4~
O
O
C
1~
g-24
INGUINAL
C
g
g-24
PARAMETRIAL
C
g
g-24
GABTROCNRMIU8
1
g
g-'m4
HEART
FIG. 2. Inguinal and parametrial post-mitochondrial adipose tissue lipoprotein lipase activity (mean+-S.E.). C=non-exercised rats; E=data collected immediately after exercise; E-24=data collected 24 hours after exercise. *p<0.05, n=8.
FIG. 3. Gastrocnemius and heart muscle heparin released lipoprotein lipase activity (mean_+S.E.). C=non-exercised rats; E=data collected immediately after exercise; E-24=data collected 24 hours after exercise. *p<0.05, n=8.
Gastrocnemius citrate synthase remained unchanged immediately after exercise but reached values significantly higher 24 hours later (p<0.05) (Fig 4). Heart citrate synthase values are not significantly changed with exercise (Fig. 4). Blood glycerol and blood glucose increased after exercise, with E greater than C and E-24 post-exercise (p<0.05). No change was observed for the plasma glucocorticoid levels (Table 2).
while no effects of exercise were apparent for parametrial fat cell lipolysis, its LPL activity was gradually suppressed in a manner similar to that reported by Barakat et al. [4]; the lowest values being observed 24 hours after the bout of exercise. The present results are thus consistent with the interpretation that one bout of high intensity exercise has site specific effects on adipose tissue metabolism. Moreover, parametrial adipose tissue LPL activity reached values about 4 fold higher than in the inguinal depot. This finding has also been reported by others [17] and stresses the possibility that each tissue has a specific physiological role. The latter specificity has also been observed during chronic exercisetraining. Indeed, recent results from our laboratory have confirmed that parametrial and retroperitoneal adipose tissues exhibit a greater metabolic response to exercise training during pregnancy than does the inguinal depot [41]. The present results appeared to be independent of food intake. For example, while adipose tissue LPL activity was decreased 24 hr after exercise, exercised and non-exercised animals had similar food intake. Similar results were also reported for the epididymal fat depot of Holtzman [4] and Wistar rats [38]. The mechanism responsible for the regional specificity observed in the response to one bout of exercise in adipose tissue lipolytic and LPL activities could occur with the action of the sympathetic innervation and blood flow. At the level of adipose tissue, a sympathetic innervation is first known to induce a local vasoconstriction along with a decreased metabolic activity; second, an accumulation of adenosine in adipose tissue; and third, a local vasodilatation particularly strong when the stimuli is intense [14,31]. Pefonnet et al. [34,35] reported that the local sympathetic stimulation is mainly responsible for the metabolic changes of adipose tis-
Metabolic Relationships Within C and E Groups The metabolic relationships between pre- and postexercise values for gastrocnemius citrate synthase activity and heart and parametrial L P L are markedly modified by acute exercise (Table 3). Gastrocnemius citrate synthase activity is negatively correlated with heart L P L before exercise and positively afterward (,o<0.05). On the other hand, it is positively correlated with parametrial L P L activity before and negatively after the exercise bout (p<0.05). DISCUSSION
Overall Effects of Exercise Results of the present study showed clearcut differences in the metabolic responses to one bout of high intensity exercise for adipose tissues of deep and subcutaneous anatomical regions. In agreement with the results reported by Oscai [33], inguinal fat cell lipolysis was higher at the end of one bout of swimming than at the beginning, values tending to come back to control 24 hours later. Plasma glycerol levels were concordant with the latter finding, i.e., values were increased after exercise and were back to pre-exercise levels 24 hours later. Inguinal adipose tissue LPL activity was however unchanged by exercise. On the other hand,
SITE S P E C I F I C E F F E C T S O F A C U T E E X E R C I S E
69 TABLE 2
EFFECTS OF EXERCISE ON BLOOD GLYCEROL, GLUCOSE AND CORTICOSTERONE Mean
C Plasma Glycerol (ttmol/dl) Serum Glucose (mg/dl) Plasma Corticosterone (ng/ml)
E
13.48-+ 2.77
19.07-+1 . 8 0
E-24
Differences
15.32-+2.88
*
131.8 -+13.2
212.7 -+76.3
133.4 -+10.8
*
170.8 -+33.3
172.9 -+50.9
194.3 -+55.4
n.s.
Values are means-+SD, n=8. C: pre-exercise; E: post-exercise; E-24:24 hr post-exercise. *E Significantly different than C and E-24 (p<0.05). n.s.: not significant. 90"
0
60 \\\\ \\\N \\\\
*d
.
30
!
! !
\\ \\ \\ N \
r..)
C
c
E
E-24
GASTROCNEMIUS
C
E
E-24
HEART
FIG. 4. Gastrocnemius and heart muscle citrate synthase activity (mean- S.E.). C = non-exercised rats; E = data coUected immediately after exercise; E-24=data collected 24 hours after exercise. *p <0.05, n=8. sue during exercise. Moreover, changes in adipose tissue blood flow during exercise are generally reported to parallel changes of its metabolism [8,9]. The latter steps of action of the sympathetic innervation could however vary with the site studied [2]. A stimuli such as exercise was reported to reduce splanchnic blood flow [39] while it increases blood flow o f subcutaneous adipose tissue as well as its lipolytic activity [10]. This observation along with the previous results indi-
cate that the sympathetic stimulation and its consequences on blood flow could not necessarily be homogeneous. Since adipose tissue is largely infiltrated by sympathetic nerve endings, differences in the metabolic responses observed in the present study could, at least partly, happen through the heterogeneity of the sympathetic nerve stimulation. The latter stimulation could induce changes in blood flow as well as local concentrations of several metabolites such as adenosine and fatty acids known to modify metabolic activities of adipose tissue. Fat losses in exercise-trained rats were reported to be similar in both the subcutaneous and the intra-abdominal adipose depot [33]. The present results, i.e., the increased lipolytic activity in the subcutaneous depot and the decreased L P L activity in the intra-abdominal depot after exercise suggest that reduction of adiposity with exercisetraining could happen through different metabolic pathways. Moreover, the lack of a relationship between individual adipose tissue L P L and lipolytic activities before and after exercise suggests that these regulatory phenomena may be independently controlled and not reciprocally related in any stoichiometric way. The muscle metabolic responses to exercise were also different for the two tissues studied. L P L activity increased only for the heart muscle, a result similar to the results reported by Oscai [33]. This finding supports the concept that heart is an important site of intra-cellular oxidation of fatty acids derived from increased lipolysis and heart LPL. The latter oxidative process is greatly involved in the energy needs of heart muscle of rodents since their cardiac myosin ATPase was reported to be much higher than other species [27]. Citrate synthase activity, on the other hand, was responsive only in the gastrocnemius muscle, its activity having a 30% increase 24 hours after the bout of exercise. Similarly, Dohm et al. [12] reported no change of skeletal muscle citrate synthase activity in rats immediately after running until exhaustion. No values were reported within the several hours following the test [12]. It is known, however, that the mitochondrial content of skeletal muscles increases rapidly in response to exercise [6]. Indeed, Booth and Holloszy [6] reported a detectable increase of plantaris citrate synthase activity as soon as 2 days after the beginning of an exercise program on treadmill in rats. These results and ours suggest that the oxidative capacity of skeletal muscle can rapidly respond to exercise when the latter is heavy work load that
70
SAVARD, SMITH, PALMER AND GREENWOOD
TABLE 3 INTERCLASSCORRELATIONSBETWEENGASTROCNEMIUS CITRATESYNTHASEACTIVITYAND HEART AND PARAMETRIAL LPL ACTIVITIES Gast. Citrate Synthase
Heart LPL Parametrial LPL
Pre-Exercise
Post-Exercise
-0.77* 0.70*
0.74* -0.72*
*p~<0.05. stresses the synthesis of the enzyme by muscle cells [6]. While the absolute amount of work performed by each rat could not be directly measured in the present study, the level of intensity of exercise was substantial. Indeed, swimming at a non-exhaustive level has been reported to at least double the oxygen consumption in rats [23]. The present swimming protocol was designed to exercise animals continuously at high intensity for two hours. Increased blood glucose concentrations such as reported in this study are usually related to heavy exercise and are thought to originate mainly from an increased glucose output from the liver [45]. In a similar fashion, Jones et al. [21] reported increased plasma glucose during heavy exercise in humans while no such observation was made with light exercise. One ought however to take into account the delay between the end of exercise and the time of sacrifice (less than 5 min); time during which liver gluconeogenesis is elevated in resting animals and contributes to increase furthermore glucose levels into the blood. Pre- and Post-Exercise Correlations
Correlations between variables that were modified by the bout of high intensity exercise were compared before and after swimming. This procedure allows us to verify qualitatively and quantitatively if the changes induced by exercise, modified the relationships between the variables. This approach, although not necessarily indicating causality, helps to clarify the manner by which each matabolic pathway is modified. Animals in a rested fed state are primarily dependent upon lipoproteins and glucose as substrates for the basal metabolic activity of tissues. The positive pre-exercise corre-
lation between gastrocnemius citrate synthase activity and parametrial LPL activity suggests that the skeletal muscle glycolytic fibers are using mainly glucose for their energetic needs while, through adipose tissue LPL, lipids are stored in the adipose tissue. Consistent with high availability of circulating substrates during rest, heart LPL is low. This low activity may be mediated by the high insulin: glucagon ratio which is characteristic of the fed state, and is thought to inhibit muscle LPL acitivity [25,26] while stimulating adipose tissue LPL. These relationships were rapidly modified after exercise. The negative correlation between post-exercise gastrocnemius citrate synthase and parametrial LPL is consistent with the interpretation that low values of adipose tissue LPL activity during exercise are related to a high oxidative metabolism of skeletal muscles, suggesting high efficiency in directing energy fuels to the working muscles. In addition, the positive correlation of gastrocnemius citrate synthase with heart LPL after exercise strengthen the interpretation that both metabolic pathways are involved in the specific supply of energy of these two muscles during exercise. One should thus expect that the latter high efficiency reached by some rats of the present study should, for others, be part of their chronic adaptation to endurance exercise. In conclusion, one bout of intense exercise appears to modify the metabolism of energy substrates in a regionally specific manner. Indeed, intra-abdominal adipose tissue metabolism responded differently to exercise than subcutaneous adipose tissue suggestive of a distinct physiological role for each adipose depot. Moreover, skeletal and heart muscle were also specifically responsive to endurance exercise. LPL activity was mainly affected in the heart while the citrate synthase responded more in the gastrocnemius muscle. ACKNOWLEDGEMENTS The authors gratefully acknowledge the expert technical assistance of Jerry Calvin and Jim Brown. We thank Dr. Marilyn Ramenofsky for determining blood corticosterone levels. We also thank Ms. Bonnie Milne from Vassar College and secretary's staff of the Department of Physical Education, University of Montreal for their typing assistance. Comments on the manuscript from Dr. Ruth Kava and Dr. David West were also very helpful. This study was supported by National Institute of Child Health and Human Development Grant HD-12637, and by the Fonds de la Recherche en Sant6 du Qurbec 830540, 840043, 850471.
REFERENCES
1. Askew, E. W., H. Barakat, C. L. Kuhl and G. L. Dohm. Response of lipogenesis and fatty acid synthetase to physical training and exhaustive exercise in rats. Lipids 10: 491-496, 1975. 2. Ballard, K. and S. Rosells. Adrenergic neurohumoral influences on circulation and lipolysis in canine omental adipose tissue. Circ Res 28: 389-395, 1971. 3. Baker, N., D. B. Learn, R. Kannan and K. R. Bruckdorfer. Comparison of lipogenic responses to dietary glucose in selected mouse adipose tissues. Ann Nutr Metal 25: 245-254, 1981. 4. Barakat, H. A., D. S. Kerr, E. B. Tapscott and G. L. Dohm. Changes in plasma lipids and lipolytic activity during recovery from exercise of untrained rats. Proc Soc Exp Biol Med 166: 162-166, 1981. 5. Bonnet, F. P., D. Rocour-Brumioul and A. Heuskin. Regional variations of adipose cell size and local cellularity in human subcutaneous fat during normal cell growth. Acta Paediatr Belg 32: 17-27, 1979.
6. Booth, F. W. and J. O. Holloszy. Cytochrome c turnover in rat skeletal muscle. J Biol Chem 252: 416-419, 1977. 7. Bukowiecki, L., J. Lupien, N. Follea, A. Paradis, D. Richard and J. Leblanc. Mechanism of enhanced lipolysis in adipose tissue of exercise-trained rats. Am J Physiol 239: E422-E429, 1980. 8. Bulow, J. Subcutaneous adipose tissue blood flow and triacyiglycerol mobilization during prolonged exercise in dogs. Pflugers Arch 392: 230-234, 1982. 9. Bulow, J. and J. Madsen. Influence of blood flow on fatty acid mobilization from lipolytically active adipose tissue. Pflugers Arch 390: 169-174, 1981. 10. Bulow, J. and E. Tondevold. Blood flow in different adipose tissue depots during prolonged exercise in dogs. Pflugers Arch 392: 235-238, 1982. 1I. Despres, J. P., C. Bouchard, L. Bukowiecki, R. Savard and J. Lupien. Morphology and metabolism of human fat cells: a reliability study, lnt J Obes 7: 231-240, 1983.
SITE SPECIFIC EFFECTS OF ACUTE EXERCISE
12. Dohm, G. L., R. L. Huston, E. W. Askew and H. L. Fleshood. Effects of exercise training, and diet on muscle citric acid cycle enzyme activity. Can J Biochem 51: 849--854, 1973. 13. Folch, J., M. Lees and G. H. Sloane Stanley. A simple method for the isolation and purification of total lipid from animal tissues. J Biol Chem 226: 497-509, 1957. 14. Fredholm, B. B.Studies on the sympathetic regulation of circulation and metabolism in isolated subcutaneous adipose tissue. Acta Physiol Scan Suppl 354: 1-47, 1970. 15. Fried, S., M. Lavau and F. X. Pi-Sunyer. Variations in glucose metabolism by fat cells from three adipose depots of the rat. Metabolism 31: 876--883, 1982. 16. Gliemann, J. Assay of insulin-like activity by the isolated fat cell method. Factors influencing the response to crystalline insulin. Diabetologia 3: 382-388, 1967. 17. Gruen, R. K. amd M. R. C. Greenwood. Adipose tissue iipoprotein lipase and glycerol release in fasted Zucker (fa/fa) rats. Am J Physiol 4: E76--E83, 1981. 18. Hietanen, E. and M. R.C. Greenwood. A comparison of lipoprotein lipase acitvity and adipocyte differenciation in growing male rats. J Lipid Res 18: 480-490, 1977. 19. Hirsch, J. and E. Gallian. Methods for the determination of adipose cell size in man and animals. J Lipid Res 9:110-119, 1968. 20. Holloszy, J. O. and F. W. Booth. Biochemical adaptations to endurance exercise in muscle. Annu Rev Physiol 38: 273-291, 1976. 21. Jones, N. J., G. J. F. Heigenhauser, A. Kuksis, C. G. Matsos, J. R. Sutton and C. J. Toews. Fat metabolism in heavy exercise. Clin Sci 59: 469-478, 1980. 22. Kannan, R. and N. Baker. Lipogenic responses to dietary glucose in selected rat adipose tissues. Indian J Biochem Biophys 18: 47-50, 1981. 23. Leblanc, J., A. Labrie, D. Lupien and D. Richard. Catecholamines and triiodo-thyronine variations and the calorigenic responses to norepinephrine in cold-adapted and exercise-trained rats. Can J Physiol Pharmacol 60: 783-787, 1982. 24. Leon, A. S., J, Conrad, D. B. Hunninghake and R. Serfass. Effects of a vigorous walking program on body composition, and carbohydrate and lipid metabolism of obese young men. Am J Clin Nutr 33: 1776-1787, 1979. 25. Lithell, H., J. Boberg, K. Hellsing, G. Lundqvist and B. Vessby. Lipoprotein-lipase activity in human skeletal muscle and adipose tissue in the fasting and fed states. Atherosclerosis 30: 89-94, 1978. 26. Lithell, H., J. Boberg, K. Hellsing, G. Lundqvist and B. Vessby. Increase of the lipoprotein-lipase activity in human skeletal muscle during clofibrate administration. Eur J Clin Invest 8: 6774, 1978. 27. Lompre, A. M., J. J. Mercadier, C. Wisnewsky, P. Bouveret, C. Pantanoli, A. D'Albis and K. Schwartz. Species- and agedependent changes in the relative amounts of cardiac myosin isoenzymes in mammals. Dev Biol 84: 286--290, 1981. 28. Lowry, O. and J. A. Passonneau. Flexible System o f Enzymatic Analysis. New York: Academic, 1972. 29. Meade, C. J. and M. Ashwell. Site differences in fat cells of New Zealand obese mice--a transplantation study. Metabolism 29: 854-858, 1980. 30. Mrosovsky, N. Body fat: What is regulated? Physiol Behav 38: 407-414, 1986.
71
31. Ngai, S. H., S. Rosell and L. Wallenberg. Nervous regulation of blood flow in the subcutaneous adipose tissue in dogs. Acta Physiol Scand 68: 397-403, 1966. 32. Pittet, P. G., D. Halliday and P. E. Bateman. Site differences in the fatty acid composition of subcutaneous adipose tissue of obese women. Br J Nutr 42: 57-60, 1979. 33. Oscai, L. B. Effect of acute exercise on tissue free fatty acids in untrained rats. Can J Physiol Pharmacol 57: 485-489, 1979. 34. Prronnet, F. and A. Imbach. Endurance and metabolic adjustments to exercise in sympathectomized (6-OHDA) rats. In: Biochemistry o f Exercise, vol 13, edited by H. G. Knuttgen, J. A. Vogel and J. Poortmans. Champaign: Human Kinetics Publishers, 1983, pp. 762-765. 35. Prronnet, F., R. Nadeau, J. de Champlain and A. Imbach. Plasma catecholamines and reponse to exercise in 6hydroxydopamine-treated dogs. Can J Physiol Pharmacol 60: 1219-1224, 1982. 36. Rebuffr-Scrive, M., J. Eldh, L. O. Hafstrom and P. Bjorntorp. Metabolism of mammary, abdominal, and femoral adipocytes in women before and after menopause. Metabolism 35: 792-797, 1986. 37. Rebuffr-Scrive, M., L. Enk, N. Crona, P. Lonnroth, L. Abrahamsson, U. Smith and P. Bjorntorp. Fat cell metabolism in different regions in women. Effect on menstrual cycle, pregnancy and lactation. J Clin Invest 75: 1-4, 1985. 38. Ranramaa, R. Acute effect of physical exercise on glycogen content and lipoprotein lipase activity in untrained diabetic rats. Med Biol 60: 139-143, 1982. 39. Rowell, L. B. Human cardiovascular adjustments to exercise and thermal stress. Physiol Rev 54: 75-159, 1974. 40. Savard, R., J. P. Desprrs, M. Marcotte and C. Bouchard. Endurance training and glucose conversion into triglycerides in human fat cells. J Appl Physiol 58: 230-235, 1985. 41. Savard, R., J. E. Palmer and M. R. C. Greenwood. The effects of exercise training on regional adipose tissue metabolism in pregnant rats. Am J Physiol 250: R837-R844, 1986. 42. Schotz, M., A. Garfinkel, J. Huebotter and J. E. Stewart. A rapid assay for lipoprotein lipase activity. J Lipid Res 28:25-31, 1969. 43. Stere, P. A. Methods ofEnzymology, vol 13, edited by J. M. Lowenstein. New York: Academic Press, 1969, p. 34. 44. Tshuchimoto, M., Y. Tokumitsu and M. Ui. Comparison of basal metabolic activities and hormonal sensitivities between mesenteric and epididymal adipocytes. Comp Biochem Physiol 82A: 377-383, 1985. 45. Wahren, J., P. Felig, G. Ahlborg and L. Jorfeldt. Glucose metabolism during leg exercise in man. J Clin Invest 50: 2715-2725, 1971. 46. Wahren, J., P. Felig, L. Hagenfeldt, R. Hendler and G. Ahlborg. Splanchnic and leg metabolism of glucose, free fatty acids and amino acids during prolonged exercise in man: Metabolic adaptation to prolonged exercise. Second International Symposium on Biochemistry of Exercise, edited by H. Howald and J. R. Poortmans. Basel: Magglingen, Birkhauser Verlag, 1973, pp. 144-153. 47. Wingfield, J. C. and D. S. Farner. The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding. Steroids 26:311-327, 1975. 48. Zucker, L. M. Fat mobilization in vitro and in vivo in the genetically obese Zucker rat "fatty." J Lipid Res 13: 234-243, 1972.